mmg_233_2014_genetics_genomicsfandomcom-20200215-history
The Hershey-Chase Experiment
Background: The Hershey-Chase experiment provided the key piece of evidence showing that DNA is the heritable material, responsible for controlling the cell, i.e. the "genetic material". After this experiment, the stage was set for Rosalind Franklin and Watson and Crick to elucidate the structure of DNA. This experiment was part of a set of experiments that won the 1969 Nobel Prize in Medicine or Physiology, shared by Max Delbrück, Salvador Luria and Alfred Hershey for their, "For their discoveries concerning the replication mechanism and the genetic structure of viruses""The Nobel Prize in Physiology or Medicine 1969". 2014. Nobelprize.org. Nobel Media AB. http://www.nobelprize.org/nobel_prizes/medicine/laureates/1969/. These three researchers coordinated a noncompetitive, and collaborative, effort called the, "Phage Group." The key Hershey-Chase experiments were performed in 1952, by Alfred Hershey and a highly gifted research assistant, Martha Chase. According to one of Martha's close friends from Cold Spring Harbor, where the work was done, Alfred Hershey managed his laboratory in a very unusual manner. There was no talking. To communicate, Hershey silently pointed at objects in order to direct Martha's work. The general consensus is that Martha's contributions to the experiment were significant, however the Noble Committee did not include her in the prizeDawson, Milly, 2003, Martha Chase dies, TheScientist.com, http://www.the-scientist.com/?articles.view/articleNo/22403/title/Martha-Chase-dies/. The Life of Martha Chase: After Martha Chase helped perform this pivotal work in genetics, she left the Hersey laboratory in Cold Spring Harbor and went to work at other research institutions in New York and California. Martha always returned to Cold Spring during the summers to attend the annual gatherings of the Phage Group. Martha entered a PhD program in 1959 at the University of Southern California. After a brief marriage marriage with a colleague scientist, Dr. Martha Chase graduated in 1964. Although the specifics are not well publicized, Chase experienced several personal misfortunes, and towards the end of the 1960s, her scientific career ended. As time went on, Martha developed a dementia-like phenotype, characterized by short-term memory loss, while her long-term term memory remained robust. After she passed away in 2003, a friend remembered her as, "A remarkable but tragic personDawson, Milly, 2003, Martha Chase dies, TheScientist.com, http://www.the-scientist.com/?articles.view/articleNo/22403/title/Martha-Chase-dies/." As a woman involved in a highly important scientific discovery that is explained in every intro biology course, her story should always be remembered. The Blender Experiments: At the time of the experiment, in 1952 and several years prior, it was not know whether DNA or protein was the material that was inherited. Because proteins were found to be significantly more complex than DNA, the general scientific consensus was that protein was the heritable material. It was known that DNA did not contain sulfur and protein often contained sulfur. It was also known that protein did not contain phosphorous and DNA did contain phosphorous. These attributes were exploited by Hershey and Chase, who used radioactive phosphorous (P-32) and radioactive sulfur (S-35) to respectively label DNA and protein of E. Coli Bacteria cultures. First, E. Coli Bacteria were grown in a growth medium containing either P-32 or S-35. Then, T2 phages were introduced to the separate bacterial colonies. The T2 phages released their genetic material into the cells. The bacterial cells were allowed to undergo the lytic cycle, releasing separate batches of progeny labeled either with S-35 or P-32. The progeny of the phages were then isolated. These progeny were then used to infect non-labeled bacterial cells. Then at just the right moment, after the phages had released their genetic material into the cell, but were still attached to the cell, a Waring Blender was used to spin the the cells with the phages, sheering off the phages from the bacteria without rupturing the bacterial cells. It was found that only P-32 entered the cells, while S-35 remained on the T2 phage coats. Inside the bacterial cells, the P-32 was detected, demonstrating that DNA was passed into the cells. This experiment was done again, allowing the viral progeny to be released from the cell. The resultant T2 progeny were measured for P-32 or S-35. Long and short, it was found that only P-32 was present in the T2 progeny, demonstrating that DNA (labeled with P-32) is the heritable (genetic) material, while sulfur, i.e. protein was not the heritable material. T2 Phages: ]] This is virus has a DNA genome, (as opposed to RNA) and specifically infects E. Coli bacteria. The virus attaches to the host bacterial cell and injects it's DNA, which uses the host cells transcriptional, and translational machinery to make more viruses. The T2 phage only undergoes a lytic life-cycleWikipedia: "Enterobacteria phage T2", http://en.wikipedia.org/wiki/Enterobacteria_phage_T2. The Scientific Process'Hershey, A.D. Chase, Martha, 1952, Independent Functions of Viral Protein and Nucelic Acid in Growth of Bacteriophage, http://scarc.library.oregonstate.edu/coll/pauling/dna/papers/hershey-independent-39-large.html Included in the seminal paper published in 1952 by Martha Chase and Alfred Hershey are several key experiments that allowed them to make the well-known, Noble-Prize worthy conclusions. I have not summarized all of these experiments. The full paper can be found here: http://scarc.library.oregonstate.edu/coll/pauling/dna/papers/hershey-independent-46-large.html ''In one of the first experiments, they used rapid changes in ionic strength of the solution containing phages to deactivate the phages via plasmolysis. Inactivation was performed with and without DNAse and with bacteria and with an antiphage "antibody". The key table is shown below and the findings are summarized from the original paper on the left, and on the right. By rapidly changing the ionic strength, and therefore osmotic pressure, and then making observations related to the integrity of the Phage membrane, this experiment demonstrated that the T2-phage has a osmotic-controlled membrane. This confirmed previous findings. By rapidly changing the ion concentration of the solution under conditions with and without DNAse they were able to monitor if escaped into the solution. When they did not perform an osmotic shock, leaving the phage membranes intact, and added DNAse, there was no P-32 labeled DNA detected. However, when they performed an osmotic shock on the phages, and treated the phages with DNAse, there was P-32 in the solution. Additionally, when These data strongly suggested that the phages had a sulfur-containing protein coat and phosphorous-containing DNA inside. The S-35 and P-32 was detected in intact phages by the antibody, but only the S-35 was detected by the antibody in the plasmolysized phages that had released their DNA. It was also found that the P-32 labeled DNA needed the S-35 labeled Phage to be inserted into the bacterial cells, and that the S-32 would bind to the bacterial cells, even when it did not have any P-32 labeled DNA. In the second experiment, they allowed the T2 phage to transmit DNA into the bacterial cells. The cells were heated treated before or after infection, at a temperature that compromised the cell membrane. DNAse was added, and the contents were centrifuged. T2 phages with phosphorous-labeled DNA were used. With the addition of DNase, followed by centrifugation, they found that the phosphorous labeled-DNA was susceptible to DNAse digestion, and present in the solution, when the cells had been heat-shocked. They also found that heat-shocking the phosphorous labeled-phages, without any bacteria present, resulted in a release of phosphorous-labeled DNA into the solution. The Blender Experiment Based on previous data by Anderson in 1951, Chase and Hershey reached the point in their study where they tried removing the differently labeled phages from the newly infected bacteria using a Warring blender. The results of the Warring Blender: The process of this experiment is summarized above. The extracellular P-32 is present for several reasons. There was inconsistency with the amount of incorporation of P-32 in the DNA of the phages, and around 5% of the supernatant remained with the sediment. The Final Experiment, Demonstration that only Progeny contained P-32: Who can summarize what happened better than Chase and Hershey: They allowed the infection of bacteria by S-35 labeled T2 phages. The phages were washed away, and then the bacteria was allowed to grow. The lytic cycle was halted with cyanide, which stopped the intracellular phage maturation, and UV-killed phages which reduced the amount of labeled progeny which would non-specifically bind to cellular material and more importantly lysed the cells. When the cells lysed, the progeny ere released. This was a fairly quick process: the UV-killed phage was added 25 minutes after the initial infection, and the cyanide was added 2 hours after the initial infection. The key table showing the data form this experiment is shown below: The cellular material was the primary contents of the sediment. The phages were the supernatant. Note the "control" of the immediate lysis (no progeny) and lysis after progeny had been formed. The sulfur content is the same. Demonstrating, a "background" sulfur content in the sediment as a result of the labeled Phage. ''' Here is the original Discussion published by Hershey and Chase: And the Image Shown on Wikipedia Today: References: